The essence of our research efforts is to design of recombinant and synthetic vaccines for the treatment of patients with cancer. At the heart of our efforts are mouse models for human cancer. Our goal is to translate findings into new treatment strategies for patients with cancer. Vaccines are traditionally thought of as preventing infectious diseases, but they may have new uses in the treatment of malignancies. In the case of cancer, it is now clear that the immune system can recognize and destroy even large quantities of established tumor. Evidence for this immune-mediated destruction comes primarily from clinical trials using interleukin-2 (IL-2), which is now an FDA approved treatment for melanoma and renal cell carcinoma. New immunotherapies based on vaccines designed to treat cancer have now been used in the clinic. These therapeutic vaccines have been clearly demonstrated to elicit anti-tumor immune responses. In a number of cases, these vaccines have also been reported to mediate the destruction of established cancer. How does one go about choosing an antigen appropriate for use in the design of a cancer vaccine? The molecular identification of the antigens present on cancers that are recognized by the immune system is central to the development of recombinant and synthetic vaccines. Because of the difficulty in predicting what peptides will be present on the cell surface, one of the most successful approaches to identifying tumor associated antigens suitable for the development of cancer vaccines starts with the anti-tumor immune response. T cells with anti-tumor reactivity are then used to screen cDNA libraries made from melanoma cell lines. Many of the tumor antigens that have been identified are tissue differentiation antigens in melanocytes and include gp100, MART-1/MelanA, tyrosinase, tyrosinase related proteins (TRP) -1/gp75 and TRP-2. Interestingly, these antigens are involved in the synthesis of melanin and give both melanocytes and deposits of melanoma tumor their dark pigment. The fact that differentiation antigens are non-mutated in most tumors has two important implications. First, expression of these tissue differentiation enzymes are shared by the great majority of melanoma nodules from the great majority of patients, and thus an off-the-shelf vaccine strategy targeting these antigens is possible (a strategy that targets a mutated antigen may have to be individualized for every mutation). Second, the non-mutated nature of these antigens suggests that immunotherapies that target these antigens could elicit auto-reactivity. One consequence of this auto-reactivity may be vitiligo, the patchy and permanent loss of pigment from the skin and hair thought to result from the auto-immune destruction of pigment cells. Vitiligo generally heralds a positive response to the intervention and has been correlated with objective shrinkage of deposits of metastatic melanoma in patients receiving high dose interleukin-2 (IL-2), a cytokine known to activate and expand T lymphocytes. Thus, there is evidence that vitiligo can be coupled with tumor regression, and that adoptive transfer of anti-tumor T cells recognizing differentiation antigens is associated with objective shrinkage of melanoma deposits. As immunogens we have used peptides and proteins made so that their sequences correspond to sequences from tumor antigens. We have also used viruses like vaccinia, influenza A, fowlpox, and adenoviruses to immunize patients with cancer. We have adapted these viruses to treat cancer by making them recombinant--that is, capable of mediating the expression of proteins that are not normally encoded by the virus. We have not yet been able to demonstrate that these recombinant viruses have clinical efficacy. However, we have shown recently that immunization of mice with a recombinant vaccinia virus encoding a melanocyte differentiation antigen (TRP-1) can both induce vitiligo and protect mice from a challenge with an experimental melanoma. One particularly exciting strategy designed to treat patients with cancer involves the use of naked nucleic acid vaccines. We have recently significantly enhanced the immunogenicity of a nucleic acid vaccine by making it self-replicating. This was accomplished by using a gene encoding an RNA replicase polyprotein derived from the Semliki Forest Virus (SFV) used in combination with a model antigen. A single intramuscular injection of a self-replicating RNA immunogen elicited antigen-specific antibody and CD8+ T cell responses at doses as low as 0.1 mg. Pre-immunization with a self-replicating RNA vector protected mice from tumor challenge and, more importantly, therapeutic immunization prolonged the survival of mice bearing established tumor. Interestingly, the self-replicating RNA vectors did not mediate the production of significantly greater quantities of the model antigen when compared with a conventional DNA vaccine in vitro. However, the enhanced efficacy in vivo correlated with a caspase-dependent apoptotic death in transfected cells. This death facilitated the uptake of apoptotic cells by dendritic cells, providing a potential mechanism for enhanced immunogenicity. Naked, non-infectious, self-replicating RNA may be an excellent candidate for the development of novel cancer vaccines.